The CD4+ subpopulation of T lymphocytes (CD4+ T cells) play a major role in the body's defense against intracellular pathogens such as pathogenic intracellular bacteria (e.g. Mycobacteria) and fungus (e.g. Candida), viruses and protozoa. In particular, the CD4+ T cells help to regulate the cell-mediated immune response to infection by such intracellular pathogens, and indeed are often referred to as “helper” T cells, by promoting a variety of immune responses from other cells (e.g. promotion and maturation of B lymphocytes and antibody responses, activation of macrophages, and enhancement of natural killer (NK) cell and CD8+ cytotoxic T cell (CTL) activity) through the release of cytokines in response to antigenic stimulation. Such antigenic stimulation is achieved by the recognition by “primed” CD4+ T cells (i.e. CD4+ T cells which have been previously exposed to an antigen to become “antigen-specific CD4+ T cells”) of an antigen presented by any antigen presenting cell expressing an appropriate class II major histocompatibility complex (MHC) protein. As mentioned above, one of the immune responses, or “effector functions”, brought about by stimulated CD4+ T cells is the activation of macrophages. Activated macrophages play a vital role in killing pathogens at sites of infection through increased phagocytic activity (i.e. activated macrophages show an increased ability to phagocytose pathogens) and other activities. The interaction between antigen-specific CD4+ T cells and macrophages forms the basis of the so-called delayed type hypersensitivity response (DTH) which has been exploited in various skin tests to determine whether a subject has been previously exposed or “sensitised” to a particular antigen (e.g. an antigen from a pathogen or an allergen). One well known type of DTH skin test is the Mantoux test which employs tuberculin as an antigen in the testing for latent tuberculosis (caused by Mycobacterium tuberculosis) in a subject or for determining whether a subject has been previously vaccinated with bacille Calmette-Guerin (BCG) tuberculosis vaccine.
The Mantoux test, and other similar delayed-type hypersensitivity skin tests such as the BioMerieux Multitest, have been widely used in the clinic and diagnostic and research laboratories for detecting antigen-specific responses, presumptively mediated by CD4+ T cells. They do, however, require a considerable amount of time to perform (i.e. in order to achieve a readable result), require the patient to return for the reading of the result, and there is considerable intra-operator variability in the application and reading of these tests. These problems combine to limit their effective use. For example, for the Mantoux test, any DTH response must be observed some 2-3 days after administration of the tuberculin antigen, which may not be feasible or desirable for the effective testing of persons living in remote locations. Moreover, assessment of skin test results are relatively subjective, leading to qualitative, rather than quantitative results.
Apart from DTH skin tests, antigen-specific CD4+ T cells can be detected, and indeed measured, by other methods. For example, using peripheral blood mononuclear cells (PBMC), antigen-specific CD4+ T cells can be measured by the method known as the lymphoproliferation assay (LPA). However, this method, which utilises the ability of CD4+ T cells to proliferate in vitro and involves detecting the incorporation of 3H-thymidine into newly synthesised DNA after 6-7 days in culture, has several limitations making it unattractive for diagnostic laboratories, namely: (i) the use of high levels of radionucleotides; (ii) a long “turnaround” time; (iii) the requirement for lengthy processing of blood samples to prepare PBMC; (iv) the dependence on carefully screened human serum, with variability from batch to batch; (v) the need for culture techniques which require a high level of training; (vi) the fact that the response can only be attributed to CD4+ T cells if CD4+ T cells are first purified; and vii) substantial inter- and intra-assay variability making standardisation between laboratories problematical. Presently, LPAs are seldom performed except in research laboratories.
Another example is a flow cytometric version of the LPA, using carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled PBMC (1). This assay has been widely used in research laboratories, and although it has the distinct advantages that it does not use radioactivity and also allows for the direct identification of responding CD4+ T cells, it still suffers from the other disadvantages of the LPA. In addition, this assay has the disadvantage that the flow cytometric analysis must be very carefully performed since the number of relevant cells can be very low, often amidst substantial background staining.
A further example is the so-called intracellular cytokine (ICC) assay. This has become a relatively widely used assay in research laboratories, and it allows for the direct detection of antigen-specific CD4+ T cells in whole blood cultures (2). This assay is, however, quite labour-intensive; requiring either a 6 hour culture with whole protein antigens (2 hours for antigen processing and presentation, plus 4 hours with brefeldin A) or 6 hour culture with peptides and brefeldin A. Further, when samples are obtained late in the day, they require a processing step late at night, or are otherwise left until the next day, which is not optimal. Moreover, a 2 hour intracellular staining step is then required after the culture, followed by flow cytometric analysis. Automation of the ICC assay is possible (3), but not generally available. Also, standardisation between laboratories is problematical.
Accordingly, there is no method for detecting antigen-specific CD4+ T cells amongst those that are presently available that are readily applicable to the clinic and diagnostic and research laboratories. Further, of those that are suitable for use in diagnostic laboratories, various problems and difficulties with them have made them unpopular. However, assays for detecting antigen-specific CD4+ T cells are not the only kind of immune response assay that have fallen from favour in diagnostic laboratories. That is, the response of lymphocytes of the immune system to the polyclonal mitogen, phytohaemagglutinin (PHA), has been previously widely used as a measure of general T cell immunocompetence (e.g. for cases of suspected primary immunodeficiency (4)); however, since this assay is based on the 3H-thymidine LPA, with the exception that the cells are harvested at day 3, it is subject to essentially the same disadvantages as the LPA.
A simpler whole blood method has been proposed in which small aliquots of anti-coagulated samples of whole blood are incubated with an antigen or PHA for 4 hours (up to 24 hours), and activation measured by up-regulation of the early T-cell activation marker antigen, CD69, on CD4+ T cells, by flow cytometry (5). This whole blood assay, with a short turnaround time and very simple cell surface readout, was an attractive approach. However, in the hands of the present applicant, it has been found that while the assay is useful for measuring polyclonal PHA responses, it is not sufficiently specific for detecting antigen-specific CD4+ T cells since an unfeasibly large number of CD4+ T cells up-regulate CD69 in response to antigen indicating a lack of specificity with this readout. Further, it was found that the analysis of results was complicated by the presence of CD69 on a small but significant subset of CD4+ T cells ex vivo.
Another example of a whole blood assay for antigen-specific CD4+ T cells that is in use is the Quantiferon® whole blood stimulation assay (CSL Limited, Melbourne, VIC, Australia). In this assay, following antigenic stimulation, the plasma is collected and assayed for IFN-γ by ELISA. However, as with the standard LPA, the response is not confirmed as being due to CD4+ T cells, since much IFN-γ is produced by CD8+ T cells, particularly in response to viral antigens, as well as by NK cells present in the cultures. At present, this assay is only licensed for detection of responses to M. tuberculosis. However, the assay has been observed to give high numbers of indeterminate results in young paediatric populations. There is a second assay for the detection of IFN-γ responses stimulated by M. tuberculosis known as the T spot TB assay approved for use in the Europe. This assay is an ELIspot based method, requiring separation of PBMC before plating out in an ELIspot plate. The assay appears to have similar specificity and sensitivity, but a lower rate of indeterminate results in paediatric and immuno-suppressed patients.
Accordingly, there remains a need for an alternative and relatively simple and cost effective method for detecting, particularly, antigen-specific CD4+ T cells, but also antigen-specific CD8+ T cells.
While investigating the kinetics of the up-regulation of the cell surface marker antigen CD25 (a transmembrane protein that is the α-chain of the interleukin-2 (IL-2) receptor) and cell division using CFSE-labelled PBMC, the present applicant found that CD25 was highly up-regulated on CD4+ T cells in response to antigen or mitogen from 24 hours onwards, preceding cell division which begins at about 48 hours onwards. At the same time, the kinetics of expression of certain co-stimulation cell surface proteins such as CD134 (OX40) and CD137 (4-1BB) was being investigated, and it was found that up-regulation of these proteins also occurred from 24 hours onwards. Genetic studies in mouse models have shown that the interaction between OX40 on CD4+ T cells and OX40 ligand (OX40L) on antigen-presenting cells is crucial for the generation of “memory” CD4+ T cells (i.e. primed CD4+ T cells that have developed into so-called memory cells able to confer immediate protection as well as the capacity to mount a more rapid and effective immune response to antigenic stimulation), and the promotion of effector CD4+ T cells survival after antigen priming. Since it had been previously reported that CD134 expression on CD4+ T cells peaks at 24-48 hours after stimulation of the T cell receptor by antigen or mitogen, and returns to baseline about 120 hours later (6), the present applicant therefore investigated whether CD25 and CD134 were co-expressed just prior to 48 hour (i.e. a timepoint that corresponded with maximal CD134 expression, but prior to the commencement of cell division), using whole blood cultures stimulated with antigens or mitogens, and might therefore be used as the basis of a relatively simple and cost effective method for the detection and/or measurement of antigen-specific CD4+ T cells. It was found that these cell surface markers for antigen-specific CD4+ T cells can be readily measured by flow cytometry after 40-44 hours incubation with antigen using sodium heparin anti-coagulated whole blood samples. Therefore, the method combines simplicity of set up (i.e. without the need for preparation of PBMC) with a simple flow cytometry read-out (i.e. without the need to permeabilise cells and detect cytokines). Further, it was found that the method has a surprisingly low background (<0.03% of CD4+ T cells), and by using the method, it has to-date been possible to readily detect specific CD4+ T cells responses to antigens contained in preparations of mycobacterial antigens, tetanus toxoid, Cytomegalovirus (CMV) lysate, vaccinia lysate and peptides of Human Immunodeficiency Virus (HIV-1). Moreover, it has been found that the method can also be used in assessing the broader responses to mitogens such as PHA and Staphylococcal enteroantigen B (SEB), as a measure of general immunocompetence. Still further, the method has application in cell sorting of antigen-specific or mitogen-activated CD4+ T cells, whereby fixation and permeabilisation steps may be avoided.